Naresh Kumar Tripathy

Comparison of phenotypic markers and neural differentiation potential of multipotent adult progenitor cells and mesenchymal stem cells

AIM To compare the phenotypic and neural differentiation potential of human bone marrow derived multipotent adult progenitor cells (MAPC) and mesenchymal stem cells (MSC). METHODS Cultures of MAPC and MSC were established in parallel from same samples of human bone marrow (n = 5). Both stem cell types were evaluated for expression of pluripotency markers including Oct-4 and Nanog by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) and expression of standard mesenchymal markers including CD14, CD34, CD44, CD45, CD73, CD90, CD105 and human leukocyte antigen (HLA)-ABC by flow cytometry. After treatment with neural induction medium both MAPC and MSC were evaluated for expression of neural proteins [neuronal filament-200 (NF-200) and glial fibrillar acidic protein (GFAP)] by immunocytochemistry and Western blotting and neural genes [NF-200, GFAP, Tau, microtubule-associated protein (MAP)-1B, MAP-2, neuron-specific enolase (NSE) and oligodendrocyte-1 (Olig-1)] by quantitative real-time-PCR. RESULTS MAPC had small trigonal shaped while MSC had elongated spindle-shaped morphology. The MAPC expressed Oct-4 and Nanog both at gene and protein levels, whereas MSC were negative for these pluripotent markers. MAPC were negative for HLA-ABC while MSC had high expression of HLA-ABC. In addition, MAPC as compared to MSC had significantly lower expression of CD44 (36.56% ± 1.92% vs 98.23% ± 0.51%), CD73 (15.11% ± 2.24% vs 98.53% ± 2.22%) and CD105 (13.81% ± 3.82% vs 95.12% ± 5.65%) (P < 0.001, for all) MAPC cultures compared to MSC cultures treated with neural induction medium had significantly higher fold change expression of NF-200 (0.64), GFAP (0.52), Tau (0.59), MAP-2 (0.72), Olig-1 (0.18) and NSE (0.29) proteins (P < 0.01 for Olig-1 and P < 0.001 for rest) as well as higher fold change expression of genes of NF-200 (1.34), GFAP (1.12), Tau (1.08), MAP-1B (0.92), MAP-2 (1.14) and NSE (0.4) (P < 0.001 for all). CONCLUSION MAPC can be differentially characterized from MSC as Oct-4 and Nanog positive stem cells with no expression of HLA-ABC and low expression of mesenchymal markers CD44, CD73 and CD105 and when compared to MSC they possess greater predilection for differentiation into neuro-ectodermal lineage.

Fetal cardiac mesenchymal stem cells express embryonal markers and exhibit differentiation into cells of all three germ layers

AIM To study the expression of embryonal markers by fetal cardiac mesenchymal stem cells (fC-MSC) and their differentiation into cells of all the germ layers. METHODS Ten independent cultures of rat fC-MSC were set up from cells derived from individual or pooled fetal hearts and studies given below were carried out at passages 3, 6, 15 and 21. The phenotypic markers CD29, CD31, CD34, CD45, CD73, CD90, CD105, CD166 and HLA-DR were analyzed by flow cytometry. The expression of embryonal markers Oct-4, Nanog, Sox-2, SSEA-1, SSEA-3, SSEA-4, TRA-1-60 and TRA 1-81 were studied by immunocytochemistry. The fC-MSC treated with specific induction medium were evaluated for their differentiation into (1) adipocytes and osteocytes (mesodermal cells) by Oil Red O and Alizarin Red staining, respectively, as well as by expression of lipoprotein lipase, PPARγ2 genes in adipocytes and osteopontin and RUNX2 genes in osteocytes by reverse-transcription polymerase chain reaction (RT-PCR); (2) neuronal (ectodermal) cells by expression of neuronal Filament-160 and Glial Fibrillar Acidic Protein by RT-PCR and immunocytochemistry; and (3) hepatocytic (endodermal) cells by expression of albumin by RT-PCR and immunocytochemistry, glycogen deposits by Periodic Acid Schiff staining and excretion of urea into the culture supernatant. RESULTS The fC-MSC expressed CD29, CD73, CD90, CD105, CD166 but lacked expression of CD31, CD34, CD45 and HLA-DR. They expressed embryonal markers, viz. Oct-4, Nanog, Sox-2, SSEA-1, SSEA-3, SSEA-4, TRA-1-81 but not TRA-1-60. On treatment with specific induction media, they differentiated into adipocytes and osteocytes, neuronal cells and hepatocytic cells. CONCLUSION Our results together suggest that fC-MSC are primitive stem cell types with a high degree of plasticity and, in addition to their suitability for cardiovascular regenerative therapy, they may have a wide spectrum of therapeutic applications in regenerative medicine.

Fetal kidney stem cells ameliorate cisplatin induced acute renal failure and promote renal angiogenesis

AIM To investigate whether fetal kidney stem cells (fKSC) ameliorate cisplatin induced acute renal failure (ARF) in rats and promote renal angiogenesis. METHODS The fKSC were isolated from rat fetuses of gestation day 16 and expanded in vitro up to 3(rd) passage. They were characterized for the expression of mesenchymal and renal progenitor markers by flow cytometry and immunocytochemistry, respectively. The in vitro differentiation of fKSC towards epithelial lineage was evaluated by the treatment with specific induction medium and their angiogenic potential by matrigel induced tube formation assay. To study the effect of fKSC in ARF, fKSC labeled with PKH26 were infused in rats with cisplatin induced ARF and, the blood and renal tissues of the rats were collected at different time points. Blood biochemical parameters were studied to evaluate renal function. Renal tissues were evaluated for renal architecture, renal cell proliferation and angiogenesis by immunohistochemistry, renal cell apoptosis by terminal deoxynucleotidyl transferase nick-end labeling assay and early expression of angiogenic molecules viz. vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF)-1α and endothelial nitric oxide synthase (eNOS) by western blot. RESULTS The fKSC expressed mesenchymal markers viz. CD29, CD44, CD73, CD90 and CD105 as well as renal progenitor markers viz. Wt1, Pax2 and Six2. They exhibited a potential to form CD31 and Von Willebrand factor expressing capillary-like structures and could be differentiated into cytokeratin (CK)18 and CK19 positive epithelial cells. Administration of fKSC in rats with ARF as compared to administration of saline alone, resulted in a significant improvement in renal function and histology on day 3 (2.33 ± 0.33 vs 3.50 ± 0.34, P < 0.05) and on day 7 (0.83 ± 0.16 vs 2.00 ± 0.25, P < 0.05). The infused PKH26 labeled fKSC were observed to engraft in damaged renal tubules and showed increased proliferation and reduced apoptosis (P < 0.05) of renal cells. The kidneys of fKSC as compared to saline treated rats had a higher capillary density on day 3 [13.30 ± 1.54 vs 7.10 ± 1.29, capillaries/high-power fields (HPF), P < 0.05], and on day 7 (21.10 ± 1.46 vs 15.00 ± 1.30, capillaries/HPF, P < 0.05). In addition, kidneys of fKSC treated rats had an up-regulation of angiogenic proteins hypoxia-inducible factor-1α, VEGF and eNOS on day 3 (P < 0.05). CONCLUSION Our study shows that fKSC ameliorate cisplatin induced ARF in rats and promote renal angiogenesis, which may be an important therapeutic mechanism of these stem cells in the disease.

Enhanced Adipogenicity of Bone Marrow Mesenchymal Stem Cells in Aplastic Anemia

Fatty bone marrow (BM) and defective hematopoiesis are a pathologic hallmark of aplastic anemia (AA). We have investigated adipogenic and osteogenic potential of BM mesenchymal stem cells (BM-MSC) in 10 AA patients (08 males and 02 females) with median age of 37 years (range: 06 to 79 years) and in the same number of age and sex matched controls. It was observed that BM-MSC of AA patients had a morphology, phenotype, and osteogenic differentiation potential similar to control subjects but adipocytes differentiated from AA BM-MSC had a higher density and larger size of lipid droplets and they expressed significantly higher levels of adiponectin and FABP4 genes and proteins as compared to control BM-MSC (P < 0.01 for both). Thus our data shows that AA BM-MSC have enhanced adipogenicity, which may have an important implication in the pathogenesis of the disease.

Fetal Kidney Cells Can Ameliorate Ischemic Acute Renal Failure in Rats through Their Anti-Inflammatory, Anti-Apoptotic and Anti-Oxidative Effects

Fetal kidney cells may contain multiple populations of kidney stem cells and thus appear to be a suitable cellular therapy for the treatment of acute renal failure (ARF) but their biological characteristics and therapeutic potential have not been adequately explored. We have culture expanded fetal kidney cells derived from rat fetal kidneys, characterized them and evaluated their therapeutic effect in an ischemia reperfusion (IR) induced rat model of ARF. The fetal kidney cells grew in culture as adherent spindle shaped/polygonal cells and expressed CD29, CD44, CD73, CD90, CD105, CD24 and CD133 markers. Administration of PKH26 labeled fetal kidney cells in ARF rats resulted in a significant decrease in the levels of blood urea nitrogen, creatinine, and neutrophil gelatinase-associated lipocalin and decreased tubular necrosis in the kidney tissues (p<0.05 for all). The injected fetal kidney cells were observed to engraft around injured tubular cells, and there was increased proliferation and decreased apoptosis of tubular cells in the kidneys (p<0.05 for both). In addition, the kidney tissues of ARF rats treated with fetal kidney cells had a higher gene expression of renotropic growth factors (VEGF-A, IGF-1, BMP-7 and bFGF) and anti-inflammatory cytokine (IL10); up regulation of anti-oxidative markers (HO-1 and NQO-1); and a lower Bax/Bcl2 ratio as compared to saline treated rats (p<0.05 for all). Our data shows that culture expanded fetal kidney cells express mesenchymal and renal progenitor markers, and ameliorate ischemic ARF predominantly by their anti-apoptotic, anti-inflammatory and anti-oxidative effects.

Altered Expression of Hematopoiesis Regulatory Molecules in Lipopolysaccharide-Induced Bone Marrow Mesenchymal Stem Cells of Patients with Aplastic Anemia

We have investigated the expression of RNA transcripts of hematopoiesis regulatory molecules, viz., macrophage inflammatory protein (MIP)-1α, tumor necrosis factor (TNF)-α, granulocyte colony-stimulating factor (G-CSF), stromal cell-derived factor (SDF)-1α, stem cell factor (SCF), and transforming growth factor (TGF)-β in lipopolysaccharide-induced bone marrow mesenchymal stem cells (BM-MSCs) and levels of their soluble forms in the culture supernatants of BM-MSCs and BM plasma of patients with acquired aplastic anemia (AA) (n = 29) and controls (n = 29). The BM-MSCs of AA patients as compared to controls had markedly lower expression of MIP-1α transcripts (p < 0.001), higher expression of TNF-α (p < 0.001), G-CSF (p < 0.001), and SDF-1α (p < 0.01) transcripts, and no difference in the expression of SCF and TGF-β transcripts. The culture supernatants of BM-MSCs and BM plasma of AA patients in comparison to controls also had lower levels of MIP-1α (p < 0.01 and p < 0.001, respectively) and higher levels of TNF-α (p < 0.05 for both) and G-CSF (p < 0.05 and p < 0.001, respectively) but with no difference in the levels of SDF-1α and SCF. The levels of TGF-β were although similar in culture supernatants of BM-MSCs of both the groups, but they were significantly lower in BM plasma of the patients than controls (p < 0.001). Our data shows that BM-MSCs of AA patients have altered expression of hematopoiesis regulatory molecules suggesting that they may have a role in the pathogenesis of the disease.